1. MICROENVIRONMENT MODELS AND THEIR USE TO STUDY AGRESSIVNESS AND TARGETED THERAPY IN B CELL MALIGNANCIES
Supervisor: assoc. prof. Marek Mráz
Chronic lymphocytic leukemia (CLL) cells and indolent lymphomas are known to be dependent on diverse microenvironmental stimuli providing them signals for survival, development, proliferation, and therapy resistance. It is known that CLL cells undergo apoptosis after cultivation in vitro, and therefore it is necessary to use models of CLL microenvironment to culture CLL cells long-term and/or to study their proliferation. Several in vitro and in vivo models meet some of the characteristics of the natural microenvironment based on the coculture of malignant cells with T-lymphocytes or stromal cell lines as supportive cell, but they also have specific limitations.
The aim of this research is to develop and use models mimicking lymphoid microenvironment to study mechanisms leading to aggressiveness in B cell malignancies and/or novel therapeutic options, e.g. drugs targeting CLL proliferation, development of resistance in long-term culture or combinatory approaches, which cannot be analyzed in experiments based on the conventional culture of CLL/lymphoma primary cells. This project will utilize models developed in the laboratory and will further optimize and modify them. The biology of CLL and responses to targeted treatment will be interrogated using the developed models. The student will utilize various functional assays, Cripr editing, RNA sequencing, genome editing, drug screening etc., with the use of primary patient’s samples and cell lines. The research might bring new insights into the microenvironmental dependencies and development of resistance to targeted therapy.
Recommended literature:
- Hoferkova E, Kadakova S, Mraz M. In Vitro and In Vivo Models of CLL-T Cell Interactions: Implications for Drug tetsing.Cancers (Basel). 2022 Jun 23;14(13):3087.
- Sharma et al. …Mraz. miR-29 Modulates CD40 Signaling in Chronic Lymphocytic Leukemia by Targeting TRAF4: an Axis Affected by BCR inhibitors. Blood 2021. https://pubmed.ncbi.nlm.nih.gov/33171493/
- Seda V. et al….Mraz. FoxO1-GAB1 Axis Regulates Homing Capacity and Tonic AKT Activity in Chronic Lymphocytic Leukemia. Blood, 2021, https://doi.org/10.1182/blood.2020008101.
- Kipps et al. Chronic lymphocytic leukaemia. Nat Rev 2017 https://pubmed.ncbi.nlm.nih.gov/28102226/
- Seda V, Mraz M. B-cell receptor signalling and its crosstalk with other pathways in normal and malignant cells. Eur J Haematol. 2015 Mar;94(3):193-205. doi: 10.1111/ejh.12427. Epub 2014 Sep 13. PMID: 25080849 Review.
Requirements on candidates:
- Motivated smart people that have the “drive” to work independently, but also willing to learn from other people in the lab and collaborate.
- Candidates should have a master’s degree in Molecular biology, Biochemistry, or similar field and have deep interest in molecular biology and cancer cell biology.
Keywords: lymphoma, microenvironment, models
2. LONG NON-CODING RNAs (lncRNAs) IN THE PATHOGENESIS OF MATURE B CELL MALIGNANCIES
Supervisor: assoc. prof. Marek Mráz
We are looking for a motivated PhD student that would like to work on the following project funded by the ERC (European Research Council) Starting grant. It has been shown that short non-coding RNAs significantly contribute the onset, progression, and therapy resistance in multiple B cell leukemias and lymphomas. We have recently described the role of miRNAs in microenvironmental interactions and aggressiveness of chronic lymphocytic leukemia and follicular lymphoma (Sharma et al…Mraz, Blood, 2021; Musilova et al…Mraz, Blood, 2018; Cerna et al…Mraz, Leukemia, 2019). However, the role of long non-coding RNAs in the pathogenesis of these diseases remains completely unknown.
In this project, the student will decipher how lncRNAs regulated BCR signaling and microenvironmental interactions in B cell malignancies. We are mainly interested in chronic lymphocytic leukaemia (CLL) and follicular lymphoma (FL). CLL is the most common leukemia in adults and FL is the most common indolent non-Hodgkin lymphoma. The clinical course of CLL/FL patients can be surprisingly variable (survival from months to decades), and both diseases still remain incurable. The course of the diseases is characterized by repeated relapses leading to the evolution of resistant disease or to the high-grade transformation to a more aggressive diffuse large B-cell lymphoma/Richter. This is associated with a poor prognosis and a high risk of early death. Number of studies showed that multiple genetic lesions are associated with CLL/FL aggressiveness or transformation; however, precise molecular mechanisms underlying these processes are largely unclear. The project aims to reveal the molecular mechanisms involving lncRNAs and/or miRNAs responsible for CLL/FL aggressiveness, especially activation of BCR signaling and B-T cell interactions. The primary samples will be analyzed on the level of protein-coding as well as non-coding genes (NGS with Illumina, preliminary data available). This will be followed by searching for the function of lncRNAs using CRISPR interference, mouse models, and molecular biology technics. This will help to better understand the disease biology and possibly to identify novel molecular targets that could be used therapeutically.
Recommended literature:
- Sharma et al. …Mraz. miR-29 Modulates CD40 Signaling in Chronic Lymphocytic Leukemia by Targeting TRAF4: an Axis Affected by BCR inhibitors. Blood 2021. https://pubmed.ncbi.nlm.nih.gov/33171493/
- Musilova et al. …Mraz. miR-150 downregulation contributes to the high-grade transformation of follicular lymphoma by upregulating FOXP1 levels . Blood. 2018 Nov 29;132(22):2389-2400. https://pubmed.ncbi.nlm.nih.gov/33786575/
- Musilova K, Mraz M. MicroRNAs in B-cell lymphomas: how a complex biology gets more complex. Leukemia. 2015 May;29(5):1004-17.
- Zeni and Mraz LncRNAs in adaptive immunity: role in physiological and pathological conditions. RNA Biol. 2021 May;18(5):619-632. https://pubmed.ncbi.nlm.nih.gov/33094664/
Requirements on candidates:
- Motivated smart people that have the “drive” to work independently, but also willing to learn from other people in the lab and collaborate.
- Candidates should have a master’s degree in Molecular biology, Biochemistry, or similar field and have deep interest in molecular biology and cancer cell biology.
Keywords: Long non-coding RNA, lymphomas, DLBCL, miRNA
3. ROLE OF FOXO1 TRANSCRIPTION FACTOR IN B-CELL MALIGNANCIES
Supervisor: assoc. prof. Marek Mráz
The project is focused on FoxO1 transcription factor and its role in the pathophysiology of chronic lymphocytic leukemia (CLL). Although the therapy of CLL has made several remarkable improvements, the disease still remains incurable. Current therapeutic approaches mainly include the use of so-called B-cell receptor signaling inhibitors (ibrutinib, idelalisib) leading to disruption of pro-survival and pro-proliferative interactions in immune niches, and the use of venetoclax, which “inhibits” BCL2 anti-apoptotic protein. We have previously described that FoxO1 protein is crucial for homing capacity of CLL cell to immune niches and tonic Akt activity supporting the survival of malignant B cells (Seda et al…Mraz, Blood, 2021). Results from the FoxO1 knockout CLL cell line suggest that FoxO1 is involved in adaptation to targeted therapy (our unpublished data). The project aims to investigate further the role of FoxO1 in microenvironmental interactions, regulation of apoptosis, and adaptation to targeted therapy. The student will use technics such as genome editing (CRISPR), CHIP seq, RNA sequencing, drug testing in vitro, primary CLL samples obtained on therapy, and functional studies with various in vitro and in vivo models. The research is also relevant for the pre-clinical development of novel drugs and their combinations (several patents submitted by the lab).
Recommended literature:
- Seda V. et al….Mraz. FoxO1-GAB1 Axis Regulates Homing Capacity and Tonic AKT Activity in Chronic Lymphocytic Leukemia. Blood, 2021, https://doi.org/10.1182/blood.2020008101.
- Ondrisova L, Mraz M. Genetic and Non-Genetic Mechanisms of Resistance to BCR Signaling Inhibitors in B Cell Malignancie. Front Oncol. 2020 Oct 26;10:591577.
- Kipps et al. Chronic lymphocytic leukaemia. Nat Rev 2017 https://pubmed.ncbi.nlm.nih.gov/28102226/
- Seda V, Mraz M. B-cell receptor signalling and its crosstalk with other pathways in normal and malignant cells. Eur J Haematol. 2015 Mar;94(3):193-205. doi: 10.1111/ejh.12427. Epub 2014 Sep 13. PMID: 25080849 Review.
Requirements on candidates:
- Motivated smart people that have the “drive” to work independently, but also willing to learn from other people in the lab and collaborate.
- Candidates should have a master’s degree in Molecular biology, Biochemistry, or similar field and have deep interest in molecular biology and cancer cell biology.
Keywords: CLL, FoxO1, targeted therapy, microenvironment, apoptosis
4. REGULATION OF BCR SIGNALLING BY DNA DAMAGE RESPONSE AND P53 PROTEIN
Supervisor: Mgr. Miroslav Boudny, Ph.D.
Consultant: assoc. prof. Marek Mráz
We are looking for a motivated PhD student that would like to work on the following project funded by the ERC (European Research Council) Starting grant. The variable clinical course of several B cell malignancies largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We showed for the first time that there is a connection between p53 pathway and regulation of BCR signalling (Cerna et al., Leukemia 2018). We described that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling. It seems that the low FOXP1 levels limit BCR signalling partially via allowing for upregulation of a CD22 cell-surface, whose intracellular part serves as a docking site for phosphatases that limit BCR activation on the cell membrane. The student will further explore the connection between DNA damage response and the BCR signalling regulation. Additionally, the p53 aberration could also affect the basal levels of CD22/phosphatases, and thus contribute to the “tonic” BCR signalling, and general aggressiveness of the B cells. In vitro studies using CRISPR technology and inducible shRNAs for p53 will be conducted. Additionally, we have collected over 100 samples obtained during the administration of chemo-immuno therapy in B-cell chronic lymphocytic leukaemia (CLL) patients, and these can be used to validate the in vitro observations.
Recommended literature:
- Cerna et al. MicroRNA miR-34a Downregulates FOXP1 During DNA Damage Response to Limit BCR Signalling in Chronic Lymphocytic Leukaemia B Cells. Leukemia, 2019, https://doi.org/10.1038/s41375-018-0230-x.
- Cerna and Mraz P53 Limits B Cell Receptor (BCR) Signalling: A New Role for miR-34a and FOXP1. Oncotarget, 2018, https://doi.org/10.18632/oncotarget.26376.
- Kipps et al. Chronic Lymphocytic Leukaemia. Nature Reviews Disease Primers, 2017, https://doi.org/10.1038/nrdp.2016.96.
Requirements on candidates:
- Master’s degree in Molecular biology, Biochemistry, or similar field of study
- Experience of working in a laboratory
- The ability of collective work as well as independent project planning
- Desire to learn new things
Keywords: BCR signalling, DNA damage response, p53
5. REGULATION OF MICROENVIRONMENTAL INTERACTIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA BY NON-CODING RNAs
Supervisor: Mgr. Miroslav Boudny, Ph.D.
Consultant: assoc. prof. Marek Mráz
Non-coding RNAs (ncRNAs) represent the largest fraction of genes in human genome (up to 100 000). Aberrant expression of ncRNAs has been observed in almost every type of cancer. The first link between ncRNA dysfunction and cancer development came from studies of microRNAs in chronic lymphocytic leukemia (CLL) where miR-15/16 deletion is the most frequent genetic aberration and directly leads to leukaemia onset. Recently, it has been recognized that a characteristic feature of CLL is that cell survival and proliferation fully depend on signals from the microenvironment, especially T-cell interactions and B-cell receptor signaling (summarized in Hoferkova et al., Cancers 2022). We have recently described for the first time that miRNAs are involved in the regulation of CLL interactions with T cells (Sharma et al., Blood 2021). However, it remains largely unknown what other ncRNAs regulate microenvironmental interactions in CLL. To study microRNAs and long non-coding RNAs involved in microenvironmental interactions, we performed their global profiling in “resting” vs “activated” CLL cells (NGS with Illumina, preliminary data available). We hypothesize that differently expressed ncRNAs are directly or indirectly involved in cell signaling induced by contact with the microenvironment and thus contribute to the regulation of CLL cell survival and proliferation. We have validated that knock-down or over-expression of several of the candidate ncRNAs affect microenvironmental interactions in CLL.
The project's goal will be to study regulation through ncRNAs and the function of selected microRNA/lncRNA in the context of the CLL microenvironment. Experimentally, the project will include techniques such as immunoblotting, qPCR, transfections, cloning, viral transductions, genome editing (RNA interference and CRISPR), luciferase assay, and RNA sequencing. We utilize cell lines as models and validate observations on primary CLL cells from patients and in mouse models.
Recommended literature:
- Kipps et al., Chronic Lymphocytic Leukaemia. Nature Reviews Disease Primers, 2017, https://doi.org/10.1038/nrdp.2016.96.
- Sharma et al., Mir-29 Modulates CD40 Signaling in Chronic Lymphocytic Leukemia by Targeting TRAF4: An Axis Affected by BCR Inhibitors. Blood, 2021, https://doi.org/10.1182/blood.2020005627.
- Hoferkova et al., In Vitro and in Vivo Models of CLL–T Cell Interactions: Implications for Drug Testing. Cancers, 2022. https://doi.org/10.3390/cancers14133087.
- Zeni and Mraz, LncRNAs in adaptive immunity: role in physiological and pathological conditions. RNA Biology, 2020. https://www.tandfonline.com/doi/full/10.1080/15476286.2020.1838783.
Requirements on candidates:
- Master’s degree in Molecular biology, Biochemistry, or similar field of study
- Experience of working in a laboratory
- The ability of collective work as well as independent project planning
- Desire to learn new things
Keywords: miRNA, CLL, T cell, microenvironment
6. Role of transcription factors in onset and progression of B-cell malignancies
Supervisor: RNDr. Josef Vecera, Ph.D.
Transcription factors (TFs) are important regulators of cell growth, development, and hematopoietic cell differentiation. Disrupting the mechanisms that are responsible for the proper function of the transcription apparatus can lead to the onset of blood cell malignancies. The abnormal function of TFs due to dysregulation or genomic aberrations are often associated with the development of leukaemias, including chronic lymphocytic leukaemia (CLL) and other B-cell lymphomas. Much evidence from the latest research shows that CLL cells have an extra deregulated chromatin structure and show an increased incidence of activated enhancer and promoter areas, allowing TFs to bind and subsequently aberrantly activate potential oncogenes. Moreover, specific post-translational modification of some TFs have been noted as a result of dysregulated signalling in the leukaemia microenvironment and this also contributes to disease progression. This project should describe the role of candidate TFs in the development and progression of B-cell malignancies with emphasis on CLL while also testing targeted therapy options, e.g. using specific inhibitors of TFs or chromatin modification regulators that are currently available or in development.
We have identified candidate TFs that might act as novel regulators of the B cell survival, proliferation, and crosstalk with other immune cells. This will be further investigated by the Ph.D. student using technics such as genome editing (CRISPR), RNA sequencing, CHIPseq (cut-and-run), the use of primary CLL samples, and functional studies with various in vitro and in vivo models. The research is also relevant for understanding resistance mechanisms to targeted therapy.
Recommended literature:
- Beekman et al. The reference epigenome and regulatory landscape of chronic lymphocytic leukemia. Nature Medicine 2018, https://pubmed.ncbi.nlm.nih.gov/29785028/
- Kipps et al. Chronic lymphocytic leukaemia. Nat Rev 2017 https://pubmed.ncbi.nlm.nih.gov/28102226/
- Seda et al. FoxO1-GAB1 Axis Regulates Homing Capacity and Tonic AKT Activity in Chronic Lymphocytic Leukemia. Blood 2021 March (epub), https://pubmed.ncbi.nlm.nih.gov/33786575/
Requirements on candidates:
- Motivated smart people that have the “drive” to work independently, but also willing to learn from other people in the lab and collaborate.
- Candidates should have a master’s degree in Molecular biology, Biochemistry, or similar field and have deep interest in molecular biology and cancer cell biology.
Keywords: CLL, transcription factor, epigenetics, microenvironment
WHAT DO WE OFFER:
- Modern laboratories, project funded by the prestigious ERC grant = high risk and high gain, state-of-the-art instrument, stable funding, competitive scholarship
- You will work in a team of young investigators that challenge some long-standing problems in the field of hematology/immunology. We do basic science, but with the objective to help patients in the future (we have access to primary samples with hem. malignancies).
WHAT WILL YOU LEARN/DO:
- How to think and work independently as a scientist
- Writing of abstracts and papers (and course in grant writing and presentation of data)
- How to present data and will attend conferences to present your research
- You will spend 1-2 months visit(s) in collaborating labs in Europe or US
- Collaboration with experts in wet lab research and bioinformatics
- Novel methods such as Next Generation Sequencing (Illumina) and genome editing (Crispr)
- How to critically analyze scientific data (regular journal clubs)
- Classical methods of molecular biology (e.g. immunoblotting, flow cytometry, qRT-PCR, cell cultures, cloning), and you will use our in vitro models for microenvironmental interactions, and artificial activation/inhibition signalling pathways to decipher the gene regulatory loops.
- You can supervise bachelor and diploma students if interested
WHO ARE WE LOOKING FOR:
- Motivated smart people that have the “drive” to work independently, but also willing to learn from other people in the lab and collaborate.
- Candidates should have a master’s degree in Molecular biology, Biochemistry, or similar field and have deep interest in molecular biology and cancer cell biology.
HOW TO APPLY:
- To apply please submit a CV by email to: marek.mraz@email.cz (Subject: PhD School) and fill in the registration form.
- The PhD will start approx. Sept 2023 (negotiable)
OTHER INFO:
The research is funded by ERC grant, and will be conducted at CEITEC MASARYK UNIVERSITY (campus Bohunice). Our laboratory extensively collaborates with the University Hospital Brno in the same campus to obtain primary samples from patients. The campus provides a vibrant, multidisciplinary and highly collaborative scientific environment. The lab is located in Brno, the second-largest city in Czech Republic that has the biggest concentration of biomedical research in the region. Brno is one of the major cultural hubs, with a vibrant and lively atmosphere housing ~60.000 students. The city has a very good public transport and plenty of interesting places to visit within the reach of trains (within small distance of several major cities such as Prague, Vienna, Bratislava, Budapest) and close to international airports.
